Characterization of human aldehyde oxidase: substrate specificities, mode of inhibition and superoxide production

哺乳动物醛氧化酶（AOXs）是存在于包括人类和啮齿类动物在内的各种哺乳动物的许多组织中的钼黄酶。不同的物种含有不同数量的AOX异构体。迄今为止，AOX对哺乳动物的生理功能尚不清楚。然而，人类AOX1由于其在I期药物代谢中公认的作用而引起越来越多的药理学兴趣。特别是，除人类以外的哺乳动物表达多种组织特异性AOX酶的原因尚不清楚。我们建立了一个密码子优化的人AOX1在大肠杆菌中的异源表达系统。这种表达系统现在使我们能够获得足够数量的活性蛋白进行动力学表征，并为位点定向诱变和结构-功能研究奠定基础。为了明确人类AOX1的生理功能，我们将对其活性位点进行详细的研究。我们计划在人类个体中鉴定底物和抑制剂结合位点周围的选定单多核苷酸多态性（snp），并分析它们对hAOX1活性的影响。我们将进一步介绍FAD位点周围的氨基酸交换，并分析其对分子内电子转移的影响。此外，我们将分析hAOX1在活性氧（ROS）产生中的作用，因为hAOX1作为一种真正的氧化酶被认为对大量内源性O2-的产生有重要贡献。hAOX1与底物和抑制剂的共晶结构将得到解决，这将有助于了解底物和抑制剂结合的机制，为未来的药物开发提供帮助。我们的研究对于确定AOX1的生理病理功能，并有助于理解AOX1在人类中的药物代谢作用尤为重要。

Mammalian aldehyde oxidases (AOXs) are molybdo-flavoenzymes which are present in many tissues in various mammalian species, including humans and rodents. Different species contain a different number of AOX isoforms. So far, the physiological function of AOX for mammals is unknown. However, human AOX1 is of increasing pharmacological interest due to its recognized role in phase I drug metabolism. In particular, the reasons why mammals other than humans express a multiplicity of tissue-specific AOX enzymes is unknown. We established a codon-optimized heterologous expression system for human AOX1 in Escherichia coli. This expression system now enables us to obtain sufficient amounts of active protein for kinetic characterizations and sets the basis for site-directed mutagenesis and structure-function studies. To define the physiological function of human AOX1 the active site will be investigated in detail. We plan to characterize selected single polynucleotide polymorphisms (SNPs) identified around the substrate and inhibitor binding site in human individuals and analyze their effect on hAOX1 activity. We will further introduce amino acid exchanges around the FAD site and will analyze the effect on intramolecular electron transfer. Further, we will analyze the role of hAOX1 in the production of reactive oxygen species (ROS), since hAOX1 as a true oxidase was suggested to significantly contribute to the production of high amounts of endogenous O2-. The co-crystal structures of hAOX1 with substrates and inhibitors will be solved and will help to understand the mechanism of substrate and inhibitor binding for future drug developments. Our studies will be particularly important to define the physiopathological functions of AOX1 and will help to understand the drug metabolizing role of AOX1 for humans in general.